The applicant's objective is to develop an alphavirus replicon particle (VRP) vaccine based on HIV subtype C clinical isolates from South Africa. The applicant has developed an initial product design incorporating the gag gene for an initial phase I trial via the HVTN, and has initiated preclinical evaluation of alternative multi-gene vaccines for a second phase 1 trial. In addition, different VRP glycoprotein coats offer possible advantages in immunogenicity, dose, and production. The applicant's proposal addresses three objectives: Objective 1: efforts related to GMP production of subtype C HIV gag VRP vaccine. This objective aims to complete development work and GMP manufacture in response to clinical hold questions received from the FDA under an IND submitted in December, 2001: a) establish new Vero cell banks and conduct cell bank adventitious agent and tumorigenicity testing, b) manufacture pilot and GMP lots and conduct release testing (adventitious agent tests) of GMP lot, c) conduct stability and immunogenicity testing of pilot and GMP lots, d) prepare and submit IND amendment. Objective 2: efforts related to GMP production and IND submission of a multi-gene subtype C HIV VRP vaccine, This objective builds on the applicant's efforts to date in the design and construction of multiple alternative multi-gene VRP constructs employing the gag, pol, env, and nef genes in various single and combined replicons, and in process improvements needed to advance the manufacturing process beyond phase I trials: a) evaluate and incorporate alternative process improvements to the manufacturing process: 1) decrease or eliminate serum from the culture media, 2) evaluate alternative formulations to improve stability, 3) evaluate alternatives to heparin affinity chromatography to increase purity, 4) evaluate process modifications that increase VRP yield and scaleability, 5) qualify new analytical test methods and initiate assay validation work; b) produce 2-3 pilot lots and transfer new manufacturing process and test methods to GMP subcontractor; manufacture, test, and release 2-3 GMP lots; implement stability program; conduct preclinical GLP toxicology and immunogenicity testing; develop clinical protocol with HVTN and prepare IND. Objective 3: efforts related to study of alternative glycoprotein coats. Produce lab-scale multi-gene VRP with existing and new glycoprotein coats, compare mouse immunogenicity leading to primate studies in a DoD collaboration.